Compounds are currently ionized in vacuum (herein defined as low pressures relative to atmospheric pressure) ion sources of a mass spectrometer by first evaporating the analyte followed by gas-phase ionization as in electron ionization or chemical ionization, or by laser ablation of the analyte either directly or in a small molecule (chemical) matrix as in matrix-assisted laser desorption/ionization (MALDI), or by use of a high velocity particle or ion as in secondary ionization mass spectrometry (SIMS) and fast atom bombardment (FAB), or by methods such as field desorption where a high voltage is placed on emitters having sharp edges or tips to generate ions, or thermospray ionization where a solution flowing into a low pressure region is heated to rapidly effect vaporization and ionization or by placing a voltage on a solution flowing from a capillary as in electrospray ionization (ESI). All of these methods require a high energy means of producing gas-phase ions for mass analysis of analyte.
The current practice for analysis of compounds which cannot be vaporized without destruction by heat is to use MALDI or ESI, and variants thereof, or the newly developed methods of inlet ionization termed laserspray ionization inlet (LSII), solvent assisted ionization inlet (SAII), and matrix assisted ionization inlet (MAII), and vacuum ionization methods termed laserspray ionization vacuum (LSIV).
MALDI is typically used with a time-of-flight (TOF) mass spectrometer and is commonly referred to as MALDI-TOF. The source region operates at very low pressure (high vacuum). MALDI uses a small molecule matrix such as 2,5-dihydroxybenzoic acid (2,5-DHB) to facilitate ionization of nonvolatile analyte but requires an expensive laser and extraction voltage, and produces mostly singly charged ions. MALDI-TOF instruments are costly and dedicated to MALDI analysis. Limitations of MALDI include high matrix related background, hot/cold spot issues leading to irreproducibility and thus are not readily applicable to extracting quantitative data. Intermediate pressure MALDI sources, operating in the milli-Torr and sub-milli-Torr pressure range, can be interfaced with instruments that are multipurpose and provide high sensitivity analysis, but these ion sources and associated lasers are also expensive. A variant of MALDI called atmospheric pressure MALDI produces ions at atmospheric pressure before entering the mass spectrometer inlet, which in the presence of the extraction voltage cause loss of ions at the inlet aperture (‘rim loss’). Atmospheric pressure MALDI sources are available that operate on instruments designed for electrospray ionization (ESI) but are less sensitive than vacuum MALDI. Because MALDI produces primarily singly charged ions, the intermediate pressure and atmospheric pressure MALDI sources interfaced with instruments having limited mass-to-charge (m/z) range for singly charged ions limits the utility of the method to compounds within the limited mass range of the instrument. Therefore, for analysis of intact, high-mass compounds such as proteins, the MALDI-TOF instrument, with unlimited mass range is required. Contrary, proteins and protein complexes can be digested by enzymes and analyzed using high performance mass spectrometers (as in e.g., ‘bottom up’, ‘shot gun proteomics’) at the expense of the intact analyte information, time, cost, and expertise. Small molecule analysis is limited by the matrix background in competition with the ionization of the desired analyte (e.g., drugs and metabolites) at any of the pressure regimes used in MALDI. MALDI requires a laser and for vacuum MALDI, sample introduction from AP to vacuum is time consuming and requires expensive instrument modifications. Typical lasers for use in MALDI use laser fluencies of generally between 2 and 60 kJ m−2. Inducing fragmentation using collision-induced dissociation (CID) of singly charged ions produces little sequence (e.g., peptides) information, especially of fragile posttranslational or other chemical modifications that frequently are lost preventing structural information of the analyte to be obtained. Newer and improved fragmentation methods such as electron transfer dissociation (ETD) and electron capture dissociation (ECD) are not applicable to singly charged analyte ions. Because of the use of a laser, MALDI is a harsher method relative to, for example, LSI (laser fluencies of generally between 40 and 150 kJ m−2 with the laser aligned in transmission geometry) or ESI limiting applications to more sturdy, less fragile molecules and rarely to the analysis of e.g., molecular non-covalent complexes, MALDI can analyze molecular complexes after chemical crosslinking the complex prior to MALDI mass analyses.
One-Dimensional (1-D) and 2-D gel electrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and agarose gels are used in protein and deoxyribonucleic acid (DNA)/ribonucleic acid (RNA) separation and purification, and which can be coupled with ESI and MALDI-MS as well as ion mobility-MS and MS/MS but indirectly by digesting the macromolecule(s) in the gel slice and subsequently solution extracting it from the gel environment. Nevertheless, MS overcomes the need for specific and expensive antibodies for detection using, for example, Western blots; many compounds do not have specific antibodies and therefore cannot be detected. More commonly applied are liquid chromatography (LC)-MS and MS/MS or ion mobility-MS approaches for additional separation. Common to both ESI and MALDI, and the methods derived from them, are ion suppression issues, problems with the presence of salt, and robustness.
MALDI operates from the solid state and is a surface method enabling molecular surface imaging approaches to determine the localization of certain analytes within a surface. A voltage is applied, frequently several kilovolts, to lift the ions from the surface and accelerate them to the analyzer. The matrix requires having sufficient absorption at the laser wavelength used to enable matrix desorption from the surface. Analyte ionization occurs in a region very near the matrix surface (<100 microns from the surface). A notable degree of chemical background associated with the desorption process creates significant background noise especially in the low mass region (<800 m/z). MALDI is therefore limited in this mass range for which applications range from drug development (clinical applications) to forensic analyses. To increase the speed, especially for imaging applications, expensive high repetition lasers can be employed to desorb/ionize more rapidly enable the measurement of summed mass spectra from ˜100 laser shots and, in case of imaging of surfaces, ions in each mass spectrum are used to determine analyte location employing respective computing programs. Thus, the mass spectra generated from ˜100 laser shots are summed into a single mass spectrum which represents one pixel in the image. Advantages of the MALDI method is that predominantly singly charged ions are produced so that interpretation is simplified, which is important for complex mixtures.
ESI is an ionization method whereby a voltage, usually several thousand volts, is placed on a capillary through which a solution is passed relative to a counter electrode which contains the inlet entrance to the vacuum of the mass spectrometer. Highly charged liquid droplets are formed in the ESI process and desolvation of these droplets leads to formation of bare ions that are analyzed by the mass spectrometer. While the MALDI method produces primarily singly charged ions, the ESI liquid introduction method produces ions of high charge states when multiple ionization sites exist on the analyte molecule. ESI methodology is not well suited for surface analysis, although a method called desorption electrospray ionization (DESI) can sample surfaces but with rather poor spatial resolution and limited in upper molecular weight range of nonvolatile compounds. A newer variation, nano-DESI, enables improved spatial resolution measurements at the expense of critical alignment and expertise. Combined methods of laser ablation of a surface with capture of the ablated material in the ESI plume can also be used to image surfaces.
Because ESI produces multiply charged ions, the method is useful with high performance mass spectrometers having limited mass range and the multiply charged ions provide improved fragmentation efficiency using e.g., CID, ECD, and ETD relative to singly charged ions making analyses on high performance mass spectrometers with limited m/z range suitable. To increase analyte charge, small amounts (frequently <10% volume) of so-called supercharging reagent can be added to the solution instead or additionally to other reagents such as acids. Detection of multiply charged ions relative to singly charged ions is more efficient, as is the ion mobility gas-phase separation of analyte ions. However, ESI requires the analyte to be in a suitable solvent to provide “sprayable” conditions. ESI is softer than MALDI making it applicable to analyzing protein complexes with suitable solvent conditions applied. ESI can be combined with “online” LC for pre-separation applicable for soluble analyte samples. This approach is not applicable for solubility restricted or insoluble analytes or where spatial and temporal resolution matters.
Numerous ionization approaches under the terminology ambient ionization have been developed to circumvent some of the problems associated with ESI and MALDI. All ambient ionization methods capable of ionizing nonvolatile compounds are variants of ESI and MALDI and while they offer advantages for certain analyses, they all increase the complexity of the ion source. None of these methods offers a simple means of rapidly introducing analyte for conversion to gas-phase ions for analysis by MS. All these methods require use of high voltage, lasers, or other sources requiring application of energy or force to the sample. Further, the current means are not well suited for automated high throughput analysis because of expense or problems associated with robustness of the methods.
New ionization methods have recently been introduced. Inlet ionization methods used in MS include laserspray ionization inlet (LSII), matrix assisted ionization inlet (MAII), and solvent assisted ionization inlet (SAII). All of these methods produce abundant highly charged ions without the use of a voltage from the solid state (MAII, LSII) or solution (SAII). Ionization occurs in a heated channel (inlet) that connects a higher pressure region (typically atmospheric pressure) and a lower pressure region (typically the first vacuum region of a mass spectrometer. In practice, the matrices or solvents disclosed for these methods require that the channel be heated to greater than 150° C. and analyte ion abundance reaches a maximum between 250 and 450° C. Ion abundances reported for these methods are not analytically useful below 150° C. The mechanism of ionization of the inlet ionization methods is purported to involve creation of droplets of matrix or solvent within the heated channel with an excess of one charge at the droplet surface and an excess of the opposite charge in the bulk of the droplet. Removal of the surface layer by for example superheating the droplets as they traverse from the high to the low pressure regions with rapid bubbling on nucleation of the droplet. However the analyte ions are formed, the ionization event occurs inside the heated channel linking a higher and a lower pressure region.
With LSII, a laser ablates the matrix with incorporated analyte, the sample, into the heated inlet where ionization occurs. In MAII, the sample is introduced physically into the heated channel producing identical ionization as LSII with the same sample. In SAII, a solvent replaces the small molecule matrix and similarly produces analyte ions when introduced into the heated channel. In all inlet ionization methods, similar to MALDI, the matrix, or solvent, is present in the sample in orders of magnitude higher molar ratio relative to the analyte.
LSII-MS is a surface method that has the potential to characterize macromolecular structures directly from their native and complex environment with high spatial resolution important in surface imaging, similar to MALDI, but producing abundant highly charged ions as long as heat can be applied to the inlet tube. Mass spectrometers with skimmer cone inlets have been retrofitted with a heatable inlet tube to produce analytically useful analyte ions for analysis by mass spectrometry. Contrary to MALDI, LSII does not require the absorption by the matrix compound at the laser wavelength. The laser can create a shockwave so that the matrix:analyte association is ablated into the heated inlet tube. LSII was introduced on high performance mass spectrometers operating at atmospheric pressure without the application of any electrical field demonstrating its usefulness for tissue analysis and surface imaging.
A requirement for all of the inlet ionization methods is a heated inlet tube linking atmospheric pressure and the vacuum of the mass spectrometer, requiring the inlet channel be heated to greater than 150° C. for analytically useful results, especially when organic matrices are used as in MAII or LSII. Most mass or ion mobility spectrometer ion sources are not equipped with such a heated inlet tube and must be retrofitted. A large number of small molecule compounds have been shown to produce multiply charged mass spectra of peptides and small proteins at inlet temperature >400° C. Even so, the matrices reported for MAII and LSII that require a hot inlet to produce ions in good yield are also sufficiently nonvolatile that they collect on ion transmission elements within the instrument causing contamination over time and loss of instrument sensitivity. More volatile matrix compounds would alleviate this problem and a few (1,4-dihydroxy-2,6-dimethoxybenzene (DHDMB), salicylamide, 3,4-dihydroxyacetophenone, mono-methylfumarate, 4-trifluoromethylphenol) were discovered that produce analytically useful gas phase analyte ions when introduced into a heated inlet channel when the channel was heated to less than 150° C. and as low as 50° C. Some of these matrices (e.g., DHDMB) produced multiply charged analyte ions when introduced to a skimmer cone inlet using a source block temperature of 150° C., or by laser ablation of the sample in vacuum. This latter vacuum method in which an inlet channel is not available and the only energy or force supplied to the sample is from the laser is called laserspray ionization vacuum (LSIV).
LSIV was introduced on vacuum sources offering advantages and disadvantages described above for MALDI (vacuum source) except that it has an additional advantage of producing multiply charged ions from analyte similar to SAII, LSII, MAII, and ESI. Conditions for LSIV operation combine the requirements of LSII and vacuum MALDI requiring sufficient absorption of the matrix at the wavelength of the laser used and in a timeframe that allows the matrix to be removed from the matrix:analyte association by desolvation which are stringent experimental conditions. Similar to MALDI, fragile gangliosides cannot be analyzed using LSIV, contrary to LSII. The most prominent LSIV matrix at intermediate pressure is 2,5-dihydroxyacetaphenone (2,5-DHAP), also used as a MALDI and LSII matrix. The most prominent LSIV matrix at low pressure (high vacuum) is 2-nitrophloroglucinol (2-nitro-benzene-1,3,5-triol, 2-NPG). In LSIV, a laser is used to desorb/ablate the sample from vacuum conditions to form multiply charged ions directly from surfaces in intermediate pressure and low pressure mass spectrometer ion sources. Without a means of supplying heat to desolvate the charged matrix:analyte particles or droplets produced by laser ablation, LSIV is limited in its ability to analyze higher molecular weight proteins. The largest protein detected to date on an intermediate pressure source is lysozyme, molecular weight 14300, with charge states identical to ESI and inlet ionization methods using the same Waters SYNAPT G2 (Quadrupole ion mobility spectrometry (IMS) TOF mass spectrometer with a 8000 m/z upper limit). With a low pressure MALDI-TOF source, LSIV produces multiply charged ions of carbonic anhydrase.
The method described herein differs from and improves the above described ionization methods. This method produces multiply charged ions by using a matrix that when exposed to sub-atmospheric pressure conditions spontaneously produces analyte ions of charge states similar to the inlet ionization methods, but without the need of a heated channel or a force that allows the matrix:analyte association to enter the gas phase. Unlike with inlet ionization, the initial ionization event occurs from the surface of the substrate upon which the matrix:analyte, the sample, is placed by exposing the sample to sub-atmospheric pressure available with any mass spectrometer. The MAIV process is spontaneous and continues without the application of a force. No external energy is necessary, so that ionization is initiated by the energy already in the system. This initial ionization event is not dependent on a heated channel, but is affected by heat, which can be above or below room temperature, applied to the substrate, and requires exposure to sub-atmospheric pressure to initiate the ionization event. Thus, simply placing the sample using an appropriate matrix compound into a vacuum ion source of a mass spectrometer initiates the ionization event that produces abundant analyte ions. Likewise, by placing the sample in sub-atmospheric pressure conditions using an atmospheric pressure ion source inlet, such as is used with ESI, using an appropriate matrix the ionization event is initiated spontaneously. In either case, heating or cooling the substrate onto which the sample is placed, using methods known to those practiced in the art, extends the compounds that spontaneously produce ions from the method described herein. This method will be referred to as matrix assisted ionization vacuum (MAIV) and matrices that produce analyte ions by this method for analysis by mass spectrometry or ion mobility are referred to as MAIV matrices. MAIV is usefulness for tissue analysis and surface imaging of such as those of endogenous and exogenous origin and examples include drugs, metabolites, pesticides, lipids, peptides, proteins, chemically or posttranslational modified peptides or proteins, protein complex, receptors, ligands, catalysts, carbohydrates, glycans, antibodies, biomarkers, and other compounds produced by synthesis, such as synthetic polymers, on mass range limited mass spectrometers. These analytes can be pure or present in biological/synthetic environments such as urine, blood, skin, tissue sections, biofilms, eatable goods, flesh, vegetable surfaces, drug pills, bacterial, microbial, artificial bone, archaeological artifacts, painting, or synthetic polymer films, and others. The production of highly charged ions directly from surfaces in a soft manner and in high abundance allows sequencing of for example peptides and proteins using for example ETD.